Early and accurate assessment of suspected acute myocardial infarction is critically dependent on the sensitive and specific detection and quantitation in blood, serum or plasma of released cardiac muscle intracellular components in order to distinguish a potentially lethal event in need of emergency measures from non-life threatening conditions such as angina and non-cardiac chest pain such as dyspepsia. Early electrocardiographic changes are neither adequately specific nor sensitive, and the medical profession has come to rely on serum biochemical markers of cardiac tissue injury for early diagnosis. Among the markers released into the circulation from heart tissue following injury or necrosis, creatine kinase MB (CK-MB) is indicative of a cardiac event and is used diagnostically to identify patients undergoing a heart attack.
Lee and Goldman (1986, Annals of Internal Medicine 105:221-233) provide a discussion of the utility of CK-MB in the diagnosis of acute myocardial infarction. Creatine kinase is a dimeric enzyme that catalyzes the transfer of high-energy phosphate groups and is found predominantly in tissues that consume large amounts of energy. The enzyme has two subunits, each of which can be either type M (for muscle) or B (for brain), thus, three combinations exist: MM, MB, and BB. The CK-MM isoenzyme is dominant in adult skeletal muscle, whereas CK-BB is found mostly in the central nervous system. In humans, heart muscle tissue creatine kinase is about 85% MM and 15% MB. In order to specifically identify muscle injury of cardiac origin, quantitation of only the CK-MB isoenzyme in circulation is desirable.
The amino acid sequence differences between the M and B isoenzymes are exploited in diagnostic tests which specifically measure the cardiac isoenzyme CK-MB. Rapid diagnostic tests employing cardiac markers including CK-MB are described, for example, in U.S. Pat. Nos. 5,604,105 and 5,290,678. These and other procedures offer the rapidity of diagnosing myocardial infarction in the emergency room setting and offer significant medical benefit for patients. Numerous other procedures for measuring CK-MB are available, such as automatic clinical analyzers, column chromatography, radioimmunoassay, and electrophoretic methods. Diagnostic tests developed to measure the level of CK-MB in bodily fluids frequently utilize CK-MB as an antigen for the preparation of antibodies used in the assay procedure, as well as purified CK-MB used as controls and calibrators in performing the assay. Assay calibrators are used to prepare a series of dilutions by which a standard curve across the operating range of an assay is prepared; assay controls are used to confirm that an assay is operating properly by ensuring that the assayed values of pre-determined samples fall within an acceptable range around their labeled values. In order for the assay to be calibrated properly, the CK-MB controls and calibrators must remain stable and in a form which is immunodetectable by the antibody or antibodies used in the assay procedure. Rapid diagnostic tests for CK-MB likewise may utilize a positive control to ensure accurate operation of the test.
Numerous CK-MB preparations from both natural and recombinant sources have been described. U.S. Pat. No. 5,496,716 describes a stabilized CK-MB composition comprising serum protein plus a CK-MB enzymatic substrate (for example, ATP or creatine) with or without a CK-MB enzymatic product (for example, ADP or phosphocreatine). Stability with a less than a 10% loss by immunoassay after 27 days at room temperature was claimed. Recombinant human CK-MB has been expressed in COS cells as a dimer (Friedman et al., 1993, Clinical Chemistry 39:1598-1601): the subunits were expressed as separate proteins then formed non-covalent dimers within the cells. Medix Biotech Inc. presently offers for sale a recombinant CK-MB product.
However, preparation of CK-MB from natural sources is a tedious procedure involving many steps including protein isolation from tissue, with its attendant hazards of infection, and protein purification, with its attendant risks of potential degradation and variable recovery, to provide an isolated CK-MB material of suitable quality and quantity for use as a calibrator or control. CK-MB prepared by recombinant methods involves the expression of separate M and B isoforms and their subsequent association to form the MB dimer which, whether performed in vitro or occurring within a host cell expressing both isoforms, will result in the formation of the MM, BB, and MB dimer; the MB dimer must be subsequently purified from the other dimers. This subsequent protein purification caries the same potential problems as described above.
Thus, there exists a need for stable CK-MB calibrators and controls that are easy to prepare and purify, and will meet the requirements of the industry. As will be evident below, a principal object of the present invention is to provide a stable CK-MB preparation for assay and other uses which comprises the CK-M subunit and the CK-B subunit on a single polypeptide chain, prepared as a recombinant construct and expressed in a bacterial expression system as a single polypeptide. A single-chain polypeptide of this invention comprising human creatine kinase M and creatine kinase B is stable, readily purified, uncontaminated with the MM or BB dimers, and has utility for the aforementioned purposes. Moreover, the product is easily produced by the skilled artisan. This ease of production maximizes the reproducibility of the products of the invention.